Compositions and methods for producing elevated and sustained ketosis

ABSTRACT

Beta-hydroxybutyrate mineral salts in combination with medium chain fatty acids or an ester thereof such as medium chain triglycerides were used to induce ketosis, achieving blood ketone levels of (2-7 mmol/L), with or without dietary restriction. The combination results in substantial improvements in metabolic biomarkers related to insulin resistance, diabetes, weight loss, and physical performance in a short period of time. Further, use of these supplements to achieve ketosis yields a significant elevation of blood ketones and reduction of blood glucose levels. Use of these substances does not adversely affect lipid profiles. By initiating rapid ketosis and accelerating the rate of ketoadaptation, this invention is useful for the avoidance of glucose withdrawal symptoms commonly experienced by individuals initiating a ketogenic diet, and minimizes the loss of lean body mass during dietary restriction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. Pat.No. 9,675,577, entitled: “Compositions and Methods for ProducingElevated and Sustained Ketosis,” scheduled to issue on Jun. 13, 2017,which is a continuation of and claims priority to U.S. Pat. No.9,138,420, entitled: “Compositions and Methods for Producing Elevatedand Sustained Ketosis,” issued on Sep. 22, 2015, which is a continuationof and claims priority to International Patent Application No.PCT/US2014/031237, filed Mar. 19, 2014 which claims priority to U.S.Provisional Patent Application No. 61/803,203, entitled: “Compositionsand Methods for Producing Elevated and Sustained Ketosis,” filed on Mar.19, 2013, and U.S. Provisional Pat. Application No. 61/926,664,entitled: “Methods of Sustaining Dietary Ketosis and Its Effects onLipid Profile,” filed Jan. 13, 2014, the contents of which are hereinincorporated by reference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant #N00014-13-1-0062 awarded by the Department of Defense, Office of NavalResearch. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to the use of ketogenic precursors to quicklyproduce elevated and sustained levels of ketone bodies in the blood andmethods for assisting the body's transition into nutritional ketosis.Specifically, the use of a combination of medium chain triglycerides(MCT) with mineral salts of beta-hydroxybutyrate (βHB) is presented toprovide an easy and accelerated method for inducing and sustainingnutritional ketosis.

BACKGROUND OF THE INVENTION

Nutritional, or therapeutic, ketosis is the physiological state ofelevated blood ketone body levels (typically above 0.5 mmol/L) resultingfrom ketogenic diets, calorie restriction, therapeutic fasting and/orsupplementation with ketogenic precursors. Ketone bodies representalternative energy substrates for both peripheral tissues and thecentral nervous system. The two most abundant and physiologicallysignificant ketone bodies are acetoacetate and β-hydroxybutyrate (βHB),while the third ketone body, acetone, is produced as a byproduct thatthe lungs breathe off. The body produces ketone bodies duringnutritional or therapeutic ketosis in the range of 2-16 mmol/L. Themetabolism of ketone bodies is associated with anticonvulsant effects,enhanced brain metabolism, neuroprotective, muscle sparing propertiesand improvement in cognitive and physical performance. Science-basedimprovements in efficiency of cellular metabolism, managed throughketone supplementation, could have beneficial impacts on physical,cognitive health, psychological health, warfighter resilience and along-term impact on health with respect to the common avoidable diseasessuch as obesity, neurodegenerative diseases, diabetes and cancer.

Under normal conditions of the standard American diet, the brain isexclusively dependent upon the metabolism of glucose to supply itsmetabolic energy. Though the brain is only 2% of bodyweight, itrepresents 25% of total glucose consumption. Ketones can replace glucoseto supply most of the brain's metabolic energy needs (>50%) duringperiods of limited glucose availability resulting fromstarvation/fasting, caloric restriction or carbohydrate restriction asin ketogenic diets. During carbohydrate deprivation, glucoseavailability decreases causing a metabolic shift towards fatty acidbeta-oxidation and the production of ketone bodies for energyhomeostasis.

Dietary carbohydrates (carbs) include simple sugars, such as table sugar(sucrose) and complex carbohydrates (starch) found in foods likepotatoes and pasta. Carbohydrate and sugar consumption have dramaticallyincreased in the last two centuries in Western societies. When sugarsand carbohydrates are consumed by humans, the pancreas secretes insulin,a hormone used to convert the sugars and carbohydrates into glucose. Theglucose is then used by the body as a fuel source. In most Westerndiets, glucose is the body's primary fuel source.

In periods of fasting, extreme exercise, and/or low carbohydrateconsumption, glucose stores in the body are rapidly used and can becomequickly depleted. Failure to replenish glucose stores as they becomedepleted causes the body to turn to an alternative method to generateenergy by creating ketone bodies. Ketone bodies can be used by everycell of the body as a replacement fuel to satisfy the body's energyneeds, including the needs of the brain. During a prolonged fast, forexample, blood ketone levels will increase to as high as 2 or 3 mmol/L.It is conventionally understood and agreed that when blood ketones riseabove 0.5 mmol/L, the heart, brain and peripheral tissues are usingketone bodies (beta hydroxybutyrate and acetoacetate) as the primaryfuel source. This condition is referred to as ketosis, or “nutritionalketosis.” This is distinguished from, and should not be confused with,diabetic or alcoholic ketoacidosis, which is the runaway accumulation ofketone bodies and associated drop in blood pH. Diabetic ketoacidosis isassociated with the absence of insulin as occurs in those suffering fromtype 1 diabetes. Ketoacidosis typically results in blood ketone levelsin excess of 25 mmol/L in combination with metabolic derangement andelectrolyte imbalance.

When in ketosis, the body essentially burns fat for fuel. This isaccomplished because fat stores in the body are utilized to create thewater soluble ketone bodies beta-hydroxybutyrate (βHB) and acetoacetate(also known as acetylacetonate). These ketone bodies are then used bythe body as its primary energy source.

The body enters a state of ketosis when it has no dietary source ofglucose or sugar and its stores of glycogen have been depleted. Thistypically occurs during fasting, exercise, and/or pursuing acarbohydrate restricted ketogenic diet. Upon transitioning into ketosis,the body begins cleaving fats into fatty acids and glycerol andtransforms the fatty acids into acetyl CoA molecules which are theneventually transformed into ketone bodies in the liver. In other words,during ketogenic metabolism in the liver, the body uses dietary andbodily fats as its primary energy source. Consequently, once in ketosis,one can easily induce loss of body fat by reducing dietary fat intakeand adjusting carbohydrate intake low enough to sustain ketosis.

Effects of Ketosis on Cognitive and Physical Performance

Performance studies in rats, mice and human subjects have shown improvedmotor function, endurance and cognitive function with ketonesupplementation. Resilience of cardiopulmonary and neurological functionunder extreme environments of oxidative stress (hyperoxia) has beenachieved in rats given ketone supplementation Many people on a ketogenicdiet report greater mental clarity, an enhanced ability to multi-task,and a more favorable and balanced mood.

Other advantages to the ketogenic diet including anti-aging and moodstabilizing effects. Recent animal studies have demonstrated superiorperformance with respect to endurance time, volume of oxygen consumed,heart rate, blood lactate levels and power output when blood ketonelevels are elevated.

Therapeutic Ketosis to Ameliorate Disease

The ketogenic diet has been established to be efficacious in treatingdrug-resistant seizure disorders. This therapeutic method is wellestablished in children and adults. The ketogenic diet has been used totreat pediatric intractable seizures since the 1920s. The diet iscurrently being investigated as treatment for a broad list of diseasestates from cardiovascular health and type II diabetes to cancer andneurological disorders such as amyotrophic lateral sclerosis (ALS) andtraumatic brain injury. The metabolic adaptations associated with aketogenic diet improve mitochondrial function, decrease reactive oxygenspecies (ROS) production, reduce inflammation and increase the activityof neurotrophic factors. Thus, using ketones to treat traumatic braininjury has also been suggested. Additionally, some studies havesuggested that certain cancer cells cannot live on ketones and thereforeinvestigation of therapeutic ketosis for cancer is underway.

As disclosed in studies relating to the effects of ketosis on cognitionand performance, medium chain triglyceride diets have been used toalleviate symptoms of Alzheimer's disease and dementias, as seen inHenderson, et al. (U.S. Pat. No. 8,426,468). As Alzheimer's disease wasattributed to decreased neuronal metabolism and reduced glucoseavailability to neurons, it was suggested that elevating ketone bodiesin these patients provides an alternative fuel source for the neurons.Lipases hydrolyze the medium chain triglycerides to medium chain fattyacids in the duodenum, permitting uptake of the medium chain fatty acidsthat are subsequently oxidized by the liver to form ketone bodies.Alternatively, Henderson, et al. suggested intravenous administration ofmedium chain triglycerides, medium chain fatty acids, or ketone bodies.The increase in blood ketone levels provides neurons with a supplementalfuel where glucose is not accessible to the neuron, such as inAlzheimer's disease. Henderson (U.S. Pat. No. 8,124,589) used oralcompositions of medium chain triglycerides to treat age-associatedmemory impairment, thereby increasing ketone body levels in the blood.Veech (U.S. Pat. No. 6,323,237) also disclosed compositions containingketone bodies for treating neuronal damage, and also found thecompositions useful for increasing cardiac efficiency, providing energyto diabetics and patients suffering from insulin resistance. Thecompositions include esters and polymers. As amyloid proteinsresponsible for Alzheimer's disease block pyruvate dehydrogenase, whichis part of glucose metabolism, ketone bodies such asD-β-3-hydroxybutarate, acetoacetate, and derivatives of these compoundsare useful fuel sources.

Ketogenic Diets and Weight Loss

A ketogenic diet is one that is high in dietary fat and low incarbohydrates with moderate levels of protein (approximately 1-2 g/kg).The classical ketogenic diet consists of a strict regimen of 4 parts fatto 1 part protein with less than 25-50 g of carbohydrates per day. Ithas been suggested that the ideal macronutrient ratio to maintain aketogenic diet is 65-85 percent of calories from fats, 10-20 percent ofcalories from proteins, and 5 percent of calories from carbohydrates.

A significant advantage of pursuing weight loss through a ketogenic dietis that a ketogenic diet may result in loss of fat stores whilemaintaining and protecting muscle mass. Some studies have suggested thatthe muscle sparing properties of a ketogenic diet result in improvementin physical performance. Athletes who maintain nutritional ketosismaintain lower insulin levels and can better utilize fatty acids andketones for fuel, effectively sparing blood glucose, which optimizes andprolongs physical and mental performance. This state is referred to asbeing “keto adapted.” Keto adaptation occurs when the body adjusts toketosis by building up the necessary fat-burning enzymes, hormone levelsare changed to accommodate ketosis, glycogen stored in muscles and liveris reduced, and the body is carrying less water.

Individuals on the standard American diet can expect to get peak fatoxidation while exercising from between 60 to 65 percent of theirmaximum oxygen consumption (VO₂ max); higher exertion levels will thendeplete glycogen stores. Keto-adapted individuals draw proportionallymore substrate from fats and ketones (sparing glycogen) and can shiftthe peak to much higher VO₂ levels and thus sustain effort for anextended duration. Transitioning to a keto-adapted state (bloodketones>0.5 mmol/L) typically requires 1 to 2 weeks with severerestriction of carbohydrates (<25 g/day) and moderate proteinrestriction (1 g/kg/day) with the balance of macronutrient from fat. Asustained physiological decrease in glucose and insulin are required forsustained hepatic ketogenesis, which is very difficult for most humans.

Vlahakos (U.S. Pat. No. 6,613,356) provides a weight-loss compositionusing n-butyrate ions from potassium butyrate or related compounds.Butyric acid stimulates receptors in the stomach that the stomach isfull and food is stagnant in the stomach. Thus, consuming butyric acidprecursors prior to eating reduces food consumption. Testing showed thecompositions improved a patient's ability to withstand rigorousexercise, improved hypercholesterolemia and hypertriglyceridemia, andreduced fatigue.

Another advantage to pursuing weight loss through a ketogenic diet isthat being in ketosis reduces hunger. Indeed, hunger is the majorbarrier that is often cited for the inability to maintain a traditionalcalorie restricted diet.

Despite the many health advantages to pursuing a ketogenic diet andmaintaining a state of nutritional ketosis, there remain significantbarriers to pursuing and maintaining a ketogenic lifestyle. One of thesebarriers is the difficulty of transitioning into a ketogenic state. Thefastest way to deplete glucose stores in the body is through fastingcombined with exercise. This is physically and emotionally demanding andis an extreme challenge even for the most motivated and displined.

Individual reports recounting the difficulties of entering ketosis whenusing a dietary approach have been widely published. In a typicalexample, an individual reported no effects on blood ketone levels evenafter four days of strict adherence to a ketogenic diet (80% fat/20%protein; 90% of maintenance calories; less than 15 grams of carbs in anygiven day). Another common experience is extreme hunger during aweeklong ketogenic diet without experiencing satiety during meals unless100-200 g of carbohydrates per day were consumed.

The ability for much larger numbers of people to utilize the significantadvantages of ketosis are severely restricted by the ability to get intoketosis. This invention opens up the potential for large numbers ofpeople to quickly and easily get into ketosis and be able to sustain aketogenic lifestyle without the physiological and emotional challengesbrought on through the process of getting into and sustaining ketosis.

Additionally, the transition into ketosis causes lethargy andlight-headedness in many, resulting in an uncomfortable physiologicaland mental state commonly referred to as the “low carb flu.” Somesuggest that these transitory symptoms may last as long as two to threeweeks. If any carbohydrates over the restrictive amount are consumed,there is an immediate shift back to glucose utilization and thetransition into ketosis must begin anew.

The symptoms associated with transitioning from a sugar andcarbohydrate-rich diet into a state of ketosis will vary according tothe physiology of the particular subject. Thus, some people mayexperience minimal discomfort as they transition into ketosis. On theother hand, however, some people may experience these symptoms to such adegree that the symptoms present what may seem to be an insurmountableimpediment to getting into ketosis and taking advantage of the positivehealth effects that can be achieved by living in a state of ketosis. Thevast majority of those who attempt to induce ketosis through somecombination of diet, fasting, and exercise will experience some of thesesymptoms.

Because the presence of blood ketones can be easily measured by a urinetest using one of many ketone test strips available on the commercialmarket, those desirous of pursuing a state of ketosis can easily measuretheir progress. Just as those on a traditional diet can weigh themselvesand gain positive feedback by measuring weight loss, those pursuing astate of ketosis can also be encouraged by measuring their blood ketonelevels. However, when transitioning into ketosis, it may take fromseveral days to two weeks or longer for any measurable increase in bloodketone levels to manifest through a urine test. This lack of measurableprogress can be yet another impediment to pursuing a state of ketosis.

Following a ketogenic diet requires eliminating substantially all sugarsand carboydrates from the diet. To someone who derives pleasure fromeating cakes, candies, breads, and other non-ketogenic foods, thenecessary modification to their diet will pose an additional hurdle topursuing nutritional ketosis. With proper education and advanceplanning, implementing a ketogenic meal plan at home is manageable formany. However, because restaurant dining is part of the social fabricfor many people, maintaining a ketogenic diet in such social settingsrequires further education and may place one in awkward social settings,providing yet another impediment to widespread implementation of aketogenic diet. Additionally, in today's society, many people frequentlytravel. For a frequent traveler to maintain a ketogenic diet willtypically require that they either carry appropriate food with them orattempt to maintain the ketogenic diet in settings where few, if any,ketogenic foods may be available.

It has been suggested that transitioning into ketosis may be aided bytaking ketogenic medical foods or exogenous supplemental ketones.However, ketogenic fats like medium chain triglyceride oil (MCT oil) aregenerally not well tolerated by the gastrointestinal system inquantities necessary to aid in inducing ketosis. Additionally, oraladministration of βHB and acetoacetate in their free acid form isexpensive and ineffective at producing sustained ketosis. One idea hasbeen to buffer the free acid form of βHB with sodium salts, but thiscauses a potentially harmful sodium overload and mineral imbalance attherapeutic levels of ketosis and is largely ineffective at preventingseizures in animal models. Ketone salts with a balance of minerals areneeded to prevent the sodium overload, but these ketone mineral saltshave not been developed or commercialized yet.

Concerns have been raised about ketogenic diets increasing totalcholesterol and triglycerides while decreasing high density lipoprotein(HDL) levels. This lipid profile is a key predictor of heart health:atherosclerotic lesions, fatty streaks and fibrous plaques in the aortaand coronary arteries. This is more limiting during adult treatment withthe ketogenic diet. Based on the broad therapeutic potential forpursuing and sustatining a ketogenic lifestyle, the need to develop anoral ketone supplement that could safely elevate blood ketone levels totherapeutic ranges of nutritional ketosis without severe dietaryrestriction and the associated side effects is greater than ever.

As such, what is needed is a composition and corresponding treatment andmaintenance method that permits the establishment of ketosis in apatient quickly, and the maintenance of ketosis with little to noperceived impact on the patient's physiology or mental comfort.

SUMMARY OF THE INVENTION

A ketogenic diet is effective at raising blood ketone levels and haspotential broad applications, but achieving the advantages of thoseapplications requires strict compliance with the diet. The presentinvention provides a strategy to elevate and sustain blood ketone bodylevels through the administration of novel combinations of ketogenicsupplements and causes a rapid and sustained elevation of blood ketoneswith a single oral administration. The invention exploits the metabolicand physiological advantages of sustained ketosis (e.g. keto-adaptation)which utilizes ketones as an alternative fuel to improve metabolichealth, physical performance and enhance disease prevention.

As such, a composition of ketone precursors is disclosed which comprisesat least one medium chain fatty acid, or an ester thereof such as amedium chain triglyceride, and a β-hydroxybutyrate ketone source orprecursor. There are numerous sources of ketones and ketogenicprecursors. Nonlimiting examples of the beta-hydroxybutyrate compoundinclude beta-hydroxybutyrate salts such as sodium beta-hydroxybutyrateand arginine beta-hydroxybutyrate, potassium beta-hydroxybutyrate,calcium beta-hydroxybutyrate, magnesium beta-hydroxybutyrate, lithiumbeta-hydroxybutyrate, lysine beta-hydroxybutyrate, histidinebeta-hydroxybutyrate, ornithine beta-hydroxybutyrate, creatinebeta-hydroxybutyrate, agmatine beta-hydroxybutyrate, citrullinebeta-hydroxybutyrate, beta-hydroxy butyrate sodium salt, beta-hydroxybutyrate potassium salt, beta-hydroxy butyrate calcium salt,beta-hydroxy butyrate magnesium salt, or a combination of salts.Nonlimiting examples of combinations of beta-hydroxybutyrate saltsinclude sodium beta-hydroxybutyrate and arginine beta-hydroxybutyrate,or beta-hydroxy butyrate sodium salt and beta-hydroxy butyrate potassiumsalt. Other β-hydroxybutyrate ketone sources include, without limitingthe scope, 1,3-butanediol, ethyl acetoacetate, and ethylbeta-hydroxybutyrate. The compounds, are optionally administered between2 grams and 50 grams, between 5 grams and 30 grams, or between 10 gramsand 20 grams. For example, the ketone compounds are optionallyadministered at 2 grams, 4 grams, 5 grams, 6 grams, 7 grams, 8 grams, 9grams, 10 grams, 11 grams, 12 grams, 13 grams, 14 grams, 15 grams, 17grams, 19 grams, 20 grams, 22 grams, 24 grams, 26 grams, 28 grams, 30grams, 32 grams, 34 grams, 36 grams, 38 grams, 40 grams, 42 grams, 44grams, 46 grams, 48 grams, or 50 grams.

In some variations of the invention, the beta-hydroxy butyrate compoundis histidine beta-hydroxybutyrate, ornithine beta-hydroxybutyrate,creatine beta-hydroxybutyrate, agmatine beta-hydroxybutyrate, orcitrulline beta-hydroxybutyrate. The compound is optionally a racemicDL-beta hydroxybutyrate or the single isomer R-beta hydroxybutyrate.

It is also contemplated that additional ketone precursors or supplementsmight be used in combination with beta hydroxybutyrate and medium chaintriglycerides. These additional ketone precursors or supplements mightinclude acetoacetate, ketone esters, and other compounds that cause arise in blood ketone levels.

As noted, many individuals, especially females, experience lethargy andlight-headedness, referred to by some as the “low carb flu,” caused byglucose withdraw in the brain and to a depletion of minerals, especiallysodium and potassium in the plasma when entering ketosis through aketogenic diet. These symptoms can be attenuated or reversed withsufficient supplementation of sodium, potassium, calcium and magnesium.Supplemental administration of minerals prevents potassium depletion viathe renal-adrenal aldosterone pathway. As such, the invention optionallyuses mineral salts of beta-hydroxybutyrate (βHB). Mineral salts of βHBare described above and include, without limiting the scope of theinvention, potassium βHB, sodium βHB, calcium βHB, magnesium βHB,lithium βHB and any other feasible non-toxic mineral salts of βHB.Organic salts of βHB include, without limiting the scope of theinvention, salts of organic bases such as arginine βHB, lysine βHB,histidine βHB, ornithine βHB, creatine βHB, agmatine βHB, and citrullineβHB. The salts may contain the racemic DL-beta hydroxybutyrate or thesingle isomer R-beta hydroxybutyrate.

Non-limiting examples and sources of the medium chain fatty acid, or anester thereof such as a medium chain triglyceride, include coconut oil,coconut milk powder, fractionated coconut oil, palm oil, palm kerneloil, caprilic acid, isolated medium chain fatty acids, such as isolatedhexanoic acid, isolated octanoic acid, isolated decanoic acid, mediumchain triglycerides either purified or in natural form such as coconutoil, and ester derivatives of the medium chain fatty acids ethoxylatedtriglyceride, enone triglyceride derivatives, aldehyde triglyceridederivatives, monoglyceride derivatives, diglyceride derivatives, andtriglyceride derivatives, and salts of the medium chain triglycerides.Ester derivatives optionally include alkyl ester derivatives, such asmethyl, ethyl, propyl, butyl, hexyl, etc. Oils may be spray dried ontosolid supports such as maltodextrin to facilitate delivery in powderform. The at least one medium chain triglyceride is optionallyadministered at between 5 grams and 50 grams, between 10 grams and 40grams, or between 15 grams and 30 grams. As a nonlimiting example, themedium chain triglyceride is administered at 5 grams, 6 grams, 7 grams,8 grams, 9 grams, 10 grams, 11 grams, 12 grams, 13 grams, 14 grams, 15grams, 17 grams, 19 grams, 20 grams, 22 grams, 24 grams, 26 grams, 28grams, 30 grams, 32 grams, 34 grams, 36 grams, 38 grams 40 grams.

The composition optionally includes at least one non-toxic mineral salt.Nonlimiting examples include the minerals Na, Mg, V, K, Cr, Mn, Co, Cu,Zn, As, Mo and Se associated with an ion of chlorine, sulfate, iodine,bromine, or other known ion in the art. Examples include sodiumchloride, zinc sulfide, potassium iodine.

The ketone precursors are preferably ingested along with nutritionalsubstrates such as free amino acids, amino acid metabolites, vitamins,minerals, electrolytes and metabolic optimizers such as NADH, solubleubiquinol, tetrahydrobiopeterin, alpha-ketoglutaric acid, carnitine,and/or alpha lipoic acid, nutritional co-factors, calciumbeta-methyl-beta-hydroxybutyrate, arginine alpha-ketoglutarate, sodiumR-alpha lipoic acid, thiamine, riboflavin, niacin, pyridoxine, ascorbicacid, citric acid, malic acid, sodium benzoate, potassium sorbate,acesulfame K, aspartame, xanthan gum, or a combination thereof.Nonlimiting examples of nutritional co-factors include R-alpha lipoicacid, acetyl-1-carnitine, ketoisocaproate, alpha-ketoglutarate,alpha-hydroxyisocaproate, creatine, branched chain amino acids (leucine,isoleucine, valine), beta-hydroxy-beta methylbutyrate (HMB), B vitamins,vitamin C, soluble ubiquinol, and carnitine that assist in mitochondrialfunction. In some variations, the supplemental mixture shall provide nomore than 400 calories per day.

The compositions are useful for weight loss and treatment of high bloodglucose or type II diabetes and can improve the user's general health ina short period of time. In another embodiment, the βHB salt/medium chaintriglyceride formula is used to facilitate weight loss, as a braintonic, to enhance athletic performance, to help prevent diseases relatedto metabolic dysfunction, mitochondrial defect, and insulin resistance,as an adjunct to a ketogenic diet, as an anti-aging supplement, andother uses associated with improved metabolic health. A combinationβHB/MCT composition is optionally administered in a range of 1:1 to 1:2mixture to elevate blood ketones to a level that would be considered astate of nutritional ketosis. Administration can be performed with orwithout dietary restriction. In some variations, the patient preferablyfollows a ketogenic diet that restricts intake of carbohydrates andprotein during the period of administration of the βHB/MCT composition.In specific embodiments, the patient restricts the dietary intake to aratio of about 65% fat, 25% protein, and 10% carbohydrates. Thetherapeutic ketosis produced herein provides a rapid and sustainedketo-adaptation as a metabolic therapy for a wide range of metabolicdisorders, and provides nutritional support for therapeutic fasting,weight loss, and performance enhancement. As such, the composition isoptionally administered once per day, twice per day, or three times perday to a subject desiring to promote and/or sustain a state of ketosis.

In an embodiment of the invention, the preferred route of administrationof the mixture of βHB salts and MCT oil is oral. The product may bedelivered as a powdered mixture, as a ready-to-drink liquid, in hard orsoft gelatin caps, as hard-pressed tablets, concentrated gels, or anyother dosage form known to those trained in the art. The product ispreferably delivered in the form of a ready-to-drink formula consistingof a mixture of sodium and potassium βHB along with coconut milk powder.The drink may be pH adjusted with citric and/or malic acid, andartificial sweetener and flavoring can be added. The drink should behomogenized and pasteurized.

Because the supplements and methods of the present invention will raisethe level of blood ketones, the subject may enjoy greater flexibility inthe diet that must be followed to maintain a state of ketosis. Thus,while consistently taking the supplement of the present invention, asubject may be able to enjoy an occasional carbohydrate or sugar “cheat”and not significantly jeopardize their ketogenic state. Indeed, becausethe present invention facilitates the quick and easy transition intoketosis, should one need to depart from a strict ketogenic diet for aday or two, getting back into ketosis can be accomplished quickly andwithout the difficult symptoms which heretofore impeded the process.

Through the consumption of the supplements of the present invention, ameasureable increase in blood ketones can often be observed within hoursof taking the supplements. This is particularly true if the subjectmaintains a ketogenic diet while taking the supplements. Thus, whereasit may take weeks to measure an increase of blood ketones following aketogenic diet alone, the utilization of the present invention willallow the increase of blood ketones to be measured quickly, therebyencouraging and motivating those pursuing a state of ketosis.

BRIEF DESCRIPTION OF THE DRAWINGS

For a fuller understanding of the invention, reference should be made tothe following detailed description, taken in connection with theaccompanying drawings, in which:

FIG. 1 is a graph depicting summary data of mean blood βHB levels of ahuman subject that received βHB salts (4% solution containing 11 gramssodium βHB and 7.1 grams potassium βHB), MCT oil, or the combination ofβHB salts and MCT oil, at 15, 30, 60, 120, 240 and 480 minutes afteroral administration.

FIG. 2 is a graph depicting mean blood βHB levels after a singleadministration of βHB salts (4% solution containing 11 grams sodium βHBand 7.1 grams potassium βHB) in a fasted 70 kg male subject on threeconsecutive days at 30, 60, 120, 240 and 480 minutes after oraladministration.

FIG. 3 is an graph depicting mean blood βHB and glucose levels after asingle administration of βHB salts (4% solution containing 11 gramssodium βHB and 7.1 grams potassium βHB) in a fasted 70 kg male subjecton 3 consecutive days at 30, 60, 120, 240 and 480 minutes afteradministration.

FIG. 4 is a graph showing that ketone supplementation does not affectlipid profile. At week 0 (prior to administration) and week 4 totalcholesterol were not significantly different from control in any of thetest substances. Statistical analysis was performed using two-wayanalysis of variance (ANOVA) with Dunnett's post hoc test, results areconsidered significant if p<0.05. Error bars represent ±the standarderror of the mean (SEM).

FIG. 5 is a graph showing that ketone supplementation does not affectlipid profile. At week 0 and week 4, HDL levels were not significantlydifferent from the control in any of the treatment groups. Two-way ANOVAwith Dunnett's post hoc test results considered significant if p<0.05.Error bars represent ±SEM.

FIG. 6 is a graph showing that ketone supplementation does not affectlipid profile. At week 0 and week 4 triglycerides were not significantlydifferent from control in any of the treatment groups. Two-way ANOVAwith Dunnett's post hoc test, results considered significant if p<0.05.Error bars represent ±SEM.

FIGS. 7(A) and (B) are graphs showing the effects of ketonesupplementation on blood ketone levels in rats. Ketone supplementationdemonstrated significant elevation of blood ketones over 4 weeks. (A)Week 0 and (B) week 1, ketone supplements were given in an oral dosageof 5 g per kg body weight. Animals given MCT oil supplementation aloneshowed significantly elevated blood ketones starting at 30 min andlasting for 8 hours; The combination of βHB salt/MCT oil significantlyelevated ketones at 4 hrs. βHB salt alone did not significantly elevateketones. Two-way ANOVA with Dunnett's post hoc test, results consideredsignificant if p<0.05. Error bars represent ±SEM.

FIGS. 8(A) and (B) are graphs showing the effects of ketonesupplementation on blood ketone levels in rats. Ketone supplementationdemonstrated significant elevation of ketones over 4 weeks. In week 2(FIG. 8(a)) and week 3 (FIG. 8(b)), the dose of ketone supplements wasincreased to 10 g/kg for both βHB salt/MCT oil and MCT oil. Blood ketonelevels were significantly elevated at 30 minutes, lasting until 12 hourswith βHB salt/MCT oil peaking at 8 hours and MCT oil peaking at 4 hours.Supplementation with MCT oil alone resulted in significantly elevatedblood ketones starting at 30 minutes and lasting for 8 hours; Treatmentwith a combination of βHB salt/MCT oil caused significantly elevatedblood ketones at 4 hours. βHB salt alone did not significantly elevateketones. Two-way ANOVA with Dunnett's post hoc test, results consideredsignificant if p<0.05. Error bars represent ±SEM.

FIG. 9 is a graph showing the effects of ketone supplementation on bloodketone levels in rats. Ketone supplementation demonstrated significantelevation of blood ketones over 4 weeks. In week 4, the dose of ketonesupplements was increased to 10 g/kg for both the βHB salt/MCT oilcombination treatment group and the MCT oil alone treatment group. Bloodketones were significantly elevated beginning at 30 minutes and lastingfor 12 hours, with βHB salt/MCT oil peaking at 8 hours and MCT oilpeaking at 4 hours. Ketone supplementation with MCT oil resulted insignificantly elevated ketones starting at 30 min and lasting for 8hours; supplementation with the combination of βHB salt and MCT oilresulted in significantly elevated ketones at 4 hours. Supplementationwith βHB salt alone did not significantly elevate ketones. Two-way ANOVAwith Dunnett's post hoc test, results considered significant if p<0.05.Error bars represent ±SEM.

FIGS. 10(A) and (B) are graphs showing the effects of ketonesupplementation in rats on blood glucose levels at (A) week 0 and (B)week 1. Two-way ANOVA with Dunnett's post hoc test, results consideredsignificant if p<0.05. Error bars represent ±SEM.

FIGS. 11(A) and (B) are graphs showing the effects of ketonesupplementation in rats on blood glucose levels at (A) week 2 and (B)week 3. Two-way ANOVA with Dunnett's post hoc test, results consideredsignificant if p<0.05. Error bars represent ±SEM.

FIG. 12 is a graph showing the effects of ketone supplementation in ratson blood glucose levels at week 4. Two-way ANOVA with Dunnett's post hoctest, results considered significant if p<0.05. Error bars represent±SEM.

FIGS. 13(A) and (B) are graphs showing the effects of ketonesupplementation in rats on (A) organ weights, and (B) liver weights. Atweek 4, harvested liver weights were significantly decreased in rats feda combination of βHB Salt and MCT oil and those fed only βHB salt,whereas liver weights were significantly increased in animalssupplemented with only MCT oil. Two-way ANOVA with Dunnett's post hoctest, results considered significant if p<0.05. Error bars represent±SEM.

FIG. 14 is a graph showing the effects of ketone supplementation in ratson change of body weight.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the following detailed description of preferred embodiments,reference is made to the accompanying drawings, which form a parthereof, and within which are shown by way of illustration specificembodiments by which the invention may be practiced. It is to beunderstood that other embodiments may be utilized and structural changesmay be made without departing from the scope of the invention.

The term “about” or “approximately” as used herein refers to beingwithin an acceptable error range for the particular value as determinedby one of ordinary skill in the art, which will depend in part on howthe value is measured or determined, i.e. the limitations of themeasurement system, i.e. the degree of precision required for aparticular purpose, such as a pharmaceutical formulation. For example,“about” can mean within 1 or more than 1 standard deviation, per thepractice in the art. Alternatively, “about” can mean a range of up to20%, preferably up to 10%, more preferably up to 5% and more preferablystill up to 1% of a given value.

Concentrations, amounts, solubilities, and other numerical data may beexpressed or presented herein in a range format. It is to be understoodthat such a range format is used merely for convenience and brevity andthus should be interpreted flexibly to include not only the numericalvalues explicitly recited as the limits of the range, but also toinclude all the individual numerical values or sub-ranges encompassedwithin that range as if each numerical value and sub-range is explicitlyrecited. As an illustration, a numerical range of “about 1 to about 5”should be interpreted to include not only the explicitly recited valuesof about 1 to about 5, but also include the individual values andsub-ranges within the indicated range. Thus, included in this numericalrange are individual values such as 2, 3, and 4 and sub-ranges such asfrom 1-3, from 2-4 and from 3-5, etc. This same principle applies toranges reciting only one numerical value. Furthermore, such aninterpretation should apply regardless of the range or thecharacteristics being described.

As used herein “beta-hydroxybutyrate,” also known as βHB or βHB, is acarboxylic acid having the general formula CH₃CH₂OHCH₂COOH which may beutilized by a patient's body as a fuel source during instances of lowglucose levels in the patient and is considered a ketone body. In thepresent invention, salt variants of beta-hydroxybutyrate are disclosed.

As used herein “patient”, means members of the animal kingdom, includingmammals, such as but not limited to, primates including humans, gorillasand monkeys; rodents, such as mice, fish, reptiles and birds. Thepatient may be any animal requiring therapy, treatment, or prophylaxis,or any animal suspected of requiring therapy, treatment, or prophylaxis.The term treatment, as used in this definition only, is intended to meanthat regiment described is continued until the underlying disease isresolved, whereas therapy requires that the regiment alleviate one ormore symptoms of the underlying disease. Prophylaxis means that regimentis undertaken to prevent a possible occurrence, such as where a highglucose or diabetes is identified. “Patient” and “subject” are usedinterchangeably herein.

“Ketosis” as used herein refers to a subject having blood ketone levelswithin the range of about 0.5 mmol/L and about 16 mmol/L in a subject.Ketosis may improve mitochondrial function, decrease reactive oxygenspecies production, reduce inflammation and increase the activity ofneurotrophic factors.

“Keto-adaptation” as used herein refers to prolonged nutritional ketosis(>1 week) to achieve a sustained nonpathological “mild ketosis” or“therapeutic ketosis.”

The term “medium chain triglycerides” (MCT) are molecules having aglycerol backbone attached to three medium chain fatty acids. Mediumchain fatty acids range from 6 to 12 carbon atoms in length. Exemplaryfatty acids are caprylic acid, also known as octanoic acid, comprising 8carbon molecules, and capric acid, also known as decanoic acid,comprising 10 carbon molecules.

The term “administration” or “administering” is used to describe theprocess in which individual ketone esters or beta-hydroxybutyrate saltsin any combination with medium chain fatty acid derivatives aredelivered to a subject. The composition may be administered in variousways including oral, intragastric, and parenteral (referring tointravenous and intra-arterial and other appropriate parenteral routes),among others. Each of these conditions may be readily treated usingother administration routes of beta-hydroxybutyrate salts in combinationwith medium chain triglycerides, derivatives, or any combination thereofto treat a disease or condition.

Administration will often depend upon the amount of compoundadministered, the number of doses, and duration of treatment. In anembodiment, multiple doses of the agent are administered. The frequencyof administration of the agent can vary depending on any of a variety offactors, such as timing of treatment from previous treatments,objectives of the treatment, i.e., weight loss or treatment of cancer orneurological disease, and the like. The duration of administration ofthe agent, e.g., the period of time over which the agent isadministered, can vary, depending on any of a variety of factors,including patient response, desired effect of treatment, etc.

The amount of the agent contacted (e.g., administered) can varyaccording to factors such as the degree of susceptibility of theindividual, the age, sex, and weight of the individual, idiosyncraticresponses of the individual, the dosimetry, and the like. Detectablyeffective amounts of the agent of the present disclosure can also varyaccording to instrument and film-related factors. Optimization of suchfactors is well within the level of skill in the art.

The “therapeutically effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. Atherapeutically effective amount of individual beta-hydroxybutyratesalts in combination with medium chain triglycerides, derivatives, orany combination thereof is that amount necessary to provide atherapeutically effective result in vivo. The amount ofbeta-hydroxybutyrate salts in combination with medium chaintriglycerides, derivatives, or any combination of beta-hydroxybutyratesalts in combination with medium chain triglycerides or derivativesthereof must be effective to achieve a response, i.e. therapeuticketosis. In accordance with the present invention, a suitable singledose size is a dose that is capable of preventing or alleviating(reducing or eliminating) a symptom in a patient when administered oneor more times over a suitable time period. One of skill in the art canreadily determine appropriate single dose sizes for systemicadministration based on the size of a mammal and the route ofadministration.

The amount of the beta-hydroxybutyrate salts in combination with mediumchain triglycerides or derivatives will depend on absorption,distribution, metabolism, and excretion rates of thebeta-hydroxybutyrate salts in combination with medium chaintriglycerides or derivatives, the particular beta-hydroxybutyrate saltsin combination with medium chain triglycerides or derivatives, themethod of administration, and the particular disorder being treated, aswell as other factors known to those of skill in the art. The doseshould be sufficient to affect a desirable response, such as atherapeutic or prophylactic response against a particular disorder orcondition, taking into account the severity of the condition to bealleviated. The compounds may be administered once, or may be dividedand administered over intervals of time. It is to be understood thatadministration may be adjusted according to individual need andprofessional judgment of a person administrating or supervising theadministration of the compounds used in the present invention.

Statistics

All data are presented as the mean±standard error of the mean (SEM). Allcalculations were performed using statistical analysis software GraphPadPRSIM™ version 6.0a. Statistical significance was defined as p<0.05. Alldata were compared to control at the applicable time points using atwo-way ANOVA with Dunnet's multiple comparisons test.

The invention presented herein details the unexpected benefit of thecombination of medium chain triglycerides with βHB salts for thesustained elevation of ketone bodies when taken orally by mammals. Thecombination of βHB salts with medium chain triglycerides disclosedherein produced blood ketone levels at a rate and peak level greaterthan what was achieved with either component alone. Unexpectedly, inlater time periods, this elevation in blood ketones is sustained at ahigher level than what would be anticipated based on the sum of datafrom the ingestion of each component separately. The combination ofmedium chain triglycerides with βHB salts allows for lower dosing of thecomponents as compared to administering the individual compounds,thereby reducing side effects and resulting in a novel blood ketoneprofile.

Fasting can have many health benefits; however, it is often accompaniedby discomfort due to hunger and lack of energy substrate (glucose) forproper brain function and depletion of sodium and potassium.Additionally it can be associated with deleterious loss of lean bodymass. This invention reduces hunger, supplies an alternative energysubstrate to the brain (ketones) and confers a protein sparing effectthat preserves skeletal muscle mass. The following examples provideevidence for the use of supplemental ketones to elevate and sustainblood levels of βHB while maintaining low and controlled blood glucose.

Example 1—A 100 kg Male Subject

All procedures were done in accordance with the University of SouthFlorida Institutional Review Board (IRB) guidelines. BHB salts and MCToil were given to human subjects as a ketogenic food supplement andblood measurements of BHB and/or glucose were taken at predeterminedtime points.

To determine the time course of ketosis, the subjects were orally giventhe test substances of a BHB salt solution (450 mL; 4%, approximately 18g), an MCT oil (30 mL, approximately 30 g), or a combination of the aBHB salt solution and an MCT oil. Blood concentrations of glucose andBHB were determined utilizing a commercially available glucose/ketonemonitoring system (Precision Xtra® blood glucose and ketone meter) atdefined time points (0, 15, 30, 60, 120, 240 and 480 minutes followingingestion of the test substances).

The subject was administered a BHB salt solution containing 4% Na⁺/K⁺BHB salts in solution, an MCT oil, or a combination of the 4% Na⁺/K⁺ BHBsalts and the MCT oil. At 15, 30, 60, 120, 240 and 480 minutes afteradministration, blood was drawn. The subject returned on days 2 and 3for additional supplements, with blood taken as on day 1. Levels of BHBin the blood were determined as described above. Administration of theNa⁺/K⁺ BHB salt solution showed that plasma levels of BHB peaked at 30to 60 minutes after administration, as seen in Table 1 and FIG. 1.

TABLE 1 Blood βHB levels (mmol/L) of a 100 kg male subject following asingle daily of oral administration of either βHB salts (4% solutioncontaining 11 grams sodium βHB and 7.1 grams potassium βHB), 30 mL ofMCT oil or 11.1 g of βHB salts + 20 mL of MCT oil at 15, 30, 60, 120,240 and 480 minutes on three consecutive days. Time Day 1 Day 2 Day 3Mean Supplementation Min mM mM mM mM BHB salt (450 mL) 0 0.2 0.4 0.10.23 15 0.5 0.8 0.8 0.70 30 0.8 0.8 1.2 0.93 60 1 0.6 1.2 0.93 120 1.20.3 0.8 0.77 240 0.4 0.1 0.3 0.27 480 0.3 0.2 0.3 0.27 MCT (30 mL) 0 0.10.3 0.2 0.20 15 0.2 0.3 0.4 0.30 30 0.4 0.8 0.4 0.53 60 0.5 0.6 0.5 0.53120 0.5 0.6 0.3 0.47 240 0.1 0.2 0.1 0.13 480 0.2 0.1 0.1 0.13 BHB salt(450 mL) + 0 0.1 0.3 0.2 0.20 MCT (30 mL) 15 0.5 0.7 0.4 0.53 30 0.7 0.81.0 0.83 60 1.3 1.2 1.5 1.33 120 0.9 1.2 1.2 1.10 240 0.4 1.0 0.5 0.63480 0.4 0.8 0.3 0.50

Administration of MCT oil showed little elevation in BHB levels.Administration of BHB salts showed higher elevation in BHB levels thanMCT oil alone. The combination of BHB salts showed further elevation inBHB levels than either BHB salts or MCT oils alone. In comparison,administration of Na⁺/K⁺ BHB salt solution or the MCT oil alone showedpeak BHB levels at 60 minutes, which remained fairly elevated through120 minutes after administration, as seen in Tables 1 and 2 and FIG. 1.Further, administration the combination of Na⁺/K⁺ BHB salt solution andMCT oil increased βHB plasma levels well above those seen by BHB saltsor MCT oil alone, as evidence of a synergistic effect between thecombination of Na⁺/K⁺ BHB salt and MCT oil. By day 3, administration ofthe combination of MCT and Na⁺/K⁺ BHB salt solution resulted in elevatedand sustained BHB plasma levels after the oral supplementation past the120 minute time point.

TABLE 2 Mean blood βHB levels of a 100 kg male subject over 3 daysfollowing a single daily oral administration of either βHB salts (4%solution containing 11 grams sodium βHB and 7.1 grams potassium βHB),MCT oil or βHB salts + MCT oil at 15, 30, 60, 120, 240 and 480 minutes.BHB Salt solution (diluted 4% BHB) MCT (1:1 ratio of C8:C10) 4% BHB salt4% BHB salt solution solution (450 mL) + Hour (450 mL) MCT (30 mL) MCT(30 mL) 0 0.23 0.20 0.20 15 0.70 0.30 0.53 30 0.93 0.53 0.83 60 0.930.53 1.33 120 0.77 0.47 1.10 240 0.27 0.13 0.63 480 0.27 0.13 0.50

Example 2—A 70 kg Male Subject

Subject (70 kg male) was instructed to fast for three days, and wasgiven a single daily oral administration of either βHB salts (4%solution containing 11 grams sodium βHB and 7.1 grams potassium βHB),MCT oil or βHB salts+MCT oil at 15, 30, 60, 120, 240 and 480 minutes on3 consecutive days. At 15, 30, 60, 120, 240 and 480 minutes afteradministration, blood was drawn. The subject returned on days 2 and 3for additional supplements, with blood taken as on day 1, as inExample 1. BHB levels were determined as discussed in example 1.Administration of the combination of MCT oil and BHB salts overconcurrent days resulted in elevated and sustained BHB levels after oraladministration, as seen in FIG. 2. Interestingly, as supplementationprogressed to day 2 and day 3, elevated levels of BHB were observedsooner after supplementation and were sustained for a considerablylonger time after administration, while peak BHB levels were consistentwith Example 1 occurred at 60 minutes after administration, as seen inTable 3.

TABLE 3 Blood βHB levels of a 70 kg male subject following a singledaily oral administration of βHB salts (4% solution containing 11 gramssodium βHB and 7.1 grams potassium βHB), at 15, 30, 60, 120, 240 and 480minutes on 3 consecutive days. Time Day 1 Day 2 Day 3 Mean FASTED + BHBsalts (450 mL) 0 0.2 0.6 1.8 0.87 30 0.7 1.8 2.7 1.73 60 1.1 2.4 4.82.77 120 0.3 1.6 3.3 1.73 240 0.4 1.2 3 1.53 480 0.6 1.6 2.4 1.53

Blood glucose levels were determined as described above. Supplementationreduced plasma levels of glucose slightly, without any glucose peaksseen through the course of supplementation, as seen in FIG. 3.Supplementation showed reduced glucose throughout the time periodanalyzed following administration of combination of MCT and BHB salts,with a lower starting glucose level on each subsequent day, as seen inTable 4.

TABLE 4 Blood glucose levels of a 70 kg male subject following a singledaily oral administration of βHB salts (4% solution containing 11 gramssodium βHB salt and 7.1 grams potassium βHB salt), or MCT oil or βHBsalts + MCT oil at 15, 30, 60, 120, 240 and 480 minutes on 3 consecutivedays. Time Day 1 Day 2 Day 3 Mean FASTED + BHB salts (450 mL) 0 100 9080 90.00 30 95 95 71 87.00 60 85 90 68 81.00 120 82 85 65 77.33 240 8084 68 77.33 480 76 80 68 74.67

Example 3—Fasting Individuals

A combination of MCT and βHB salts, seen in the examples 1 and 2, wasorally administered to fasting individuals to accelerate the inductionof ketosis and to study the impact of the supplement in short-termsevere caloric restriction, short term fasting or intermittent fasting(e.g., alternate day fasting). The supplement provided no more than 400calories per day and the length of time of the various restrictions andfasts were between 2 and 7 days. Rapid induction of ketosis wasassociated with improved compliance with caloric restriction andfasting, presumably by reducing hunger and “glucose withdrawal” symptoms(e.g., brain fog).

Caloric restriction and/or fasting resulted in substantial improvementsin metabolic biomarkers related to inflammation and insulin resistance,thereby permitting individuals to lose weight and improve general healthin a short period of time. Individuals with high blood glucose or typeII diabetes may receive particular benefits from this supplement as ithas the potential to cause a lasting improvemenst in insulin sensitivityand overall metabolic health (e.g., improved glucose and lipidprofiles). This invention is useful for initiating and sustaining aketogenic lifestyle by helping a subject get into ketosis quickly. Theutilization of the supplement in conjunction with fasting accelerates asubject's ability to induce ketosis and is accompanied by a minimumdegree of physical and mental discomfort and a minimal amount of loss oflean body mass.

Example 4—Adult Male Sprague-Dawley Rats

Subject rats (n=74), 275-325 grams, were randomly assigned to a controlgroup (no supplementation) or to one of three ketone precursor groups:medium chain triglyceride (MCT) oil (MO), mineral (Na+/K+) salt ofβ-hydroxybutyrate (βHB) (SO), or SO+MO 1:1 combination (SM). The ketonemineral salt was developed and synthesized by Dr. Patrick Arnold fromPrototype Nutrition. Pure pharmaceutical grade MCT oil was purchasedfrom Now Foods (Bloomingdale, Ill.). SM was mixed at a 1:1 ratio.

Rats were supplemented with medium chain triglyceride (MCT) oil (MO), ora mineral (Na+/K+) salt of β-hydroxybutyrate (βHB) (SO), or SO+MO 1:1mixture (SM) and force fed a daily at a dose of 5 g/kg body weight ondays 1-14, followed by a 10 g/kg dose on days 15-28 rats Supplementationwas given between 10 am and 1 pm daily to eliminate variance based onregular eating patterns. Diets were not restricted for this study.

Effects of ketone supplementation on blood glucose, ketones, and lipids:

Once a week, animals were fasted for 4 hours prior to intragastricgavage of the appropriate ketone supplement. The four hour foodrestriction was used to eliminate normal variance in blood glucose andketone levels due to food consumption. Whole blood samples (10 μL) wereacquired from the saphenous vein of the subjects for analysis of glucoseand βHB utilizing the commercially available glucose and ketonemonitoring system Precision Xtra™ (Abbott Laboratories, Abbott Park,Ill.) at time intervals of 0, 0.5, 1, 4, 8, and 12 hours after ketoneprecursor supplements were administered, or until βHB returned tobaseline. On Day 0 (Week 0) and Day 28 (Week 4), whole blood samples (10μL) were acquired for analysis of total cholesterol, HDL, andtriglycerides for a lipid panel utilizing the commercially availablehome cholesterol analyzer Cardio Chek™ (Polymer Technology Systems,Inc., Indianapolis, Ind.) at time 0. Animals were weighed once per weekto maintain accurate dosage using the Mittler Toledo SB16001 scales.

Dietary ketone supplementation does not affect lipid profile

Total Cholesterol and HDL, taken at week 0 and again at week 4, showed areduction in all samples, including the control, as seen in FIGS. 4 and5. Analysis showed the levels in all supplements were not significantlydifferent from control after a 4-week chronic dosage of ketonesupplements. Triglyceride levels were mildly elevated at week 4 in thecontrol, βHB/MCT sample, or βHB salt sample, with little change inlevels of MCT sample, as seen in FIG. 6. However, none of the levelswere significantly different from control after a 4-week chronic dosageof ketone supplements.

Ketone supplementation causes rapid and sustained elevation of βHB

Over the 28-day experiment, ketone supplments significantly elevatedblood ketone levels without dietary restriction, as seen in FIGS. 7(A)through 9. The 5 g/kg dose for days 1-14 showed rapid elevation for theMCT oil at about 30 minutes to one hour with maximum effect at 60minutes and sustained significant elevation until 8 hours. By weeks 2and 3, when the dosage of MCT was increased to 10 g/kg, the βHB levelsincreased to a maximum level at 4 hours, followed by dropping levels ofβHB, however BHB levels were still elevated in comparison to the controlgroup, as seen in FIGS. 8(A) and (B). By week 4, the MCT oil beganhaving an earlier effect in elevated BHB levels, with βHB levels furtherelevated at 30 minutes and levels spiking at 1 hour, as seen in FIG. 9.However, βHB levels at 4 and 8 hours appear to remain at about the sameas BHB levels in weeks 2 and 3.

The 1:1 BHB salt/MCT oil combination (SM) at 5 g/kg showed significantelevation after 4 hours, but BHB levels dropped and were no longersignificant at 8 hours in weeks 0 and 1. In week 2, SM was administeredat 10 g/kg and started to show mild elevation earlier, which was foundsignificant, as seen in FIG. 8(A). Further βHB levels were elevated at 8hours beyond what was seen in weeks 0 and 1, to levels similar to MO. Byweek 3, the SM showed elevated βHB levels starting at 30 minutes, thoughless dramatic than MO, as seen in FIG. 8(B). Further, the βHB levelswere elevated at 1 and 4 hours, which was not apparent at weeks 0through 3, and the βHB levels at 8 hours were slightly depressed. Thatsaid, βHB levels were increased to significant levels from 30 minutes to8 hours after supplementation. By week 4 of SM supplementation, βHBlevels were elevated at 8 hours, similar to βHB levels seen with MOsupplementation, as seen in FIG. 9.

SO supplementation did not show significant elevation at any time point.With dose escalation to 10 g/kg in groups Control, MO, SO, and SM, fordays 14-28, both MO and SM were elevated at 30 min and remainedsignificantly elevated for up to 12 hours, shifting their peak times toMO at 4 hours and SM at 8 hours. SO did not show significant elevationat any time points even with escalated dosage.

Ketone supplementation causes rapid reduction in blood glucose.

SO supplementation did not show any significant difference compared tocontrol at week 0, whereas MO and SM reduced glucose through 4 hoursafter supplementation, seen in FIGS. 10(A) and (B). However, SO beganreducing glucose by 8 hours at week 1. By week 2 onward, with anincrease in supplementation dosage to 10 g/kg, SO, MO and SM had reducedglucose levels at 30 minutes through 8 hours, with normalization toabout control levels by 12 hours, as seen in FIGS. 11 through 12.

The effects of ketone supplementation on organ weight:

At the end of 4 weeks, rats were force fed at time 0 then sacrificed byCO₂ between 4-8 hours, which were determined to be peak BHB levelelevation. Brain, lungs, liver, kidneys, spleen and heart were harvestand weighed using AWS-1000 1 kg portable digital scale (AWS, Charleston,S.C.). Organs were then either flash frozen in liquid nitrogen orpreserved in paraformaldehyde for future analysis.

At the end of 4 weeks, livers were harvested and weighed per subjectrats. SO and SM ketone supplements significantly decreased the weight ofthe liver. MO significantly increased the weight of livers in thesubjects, as seen in FIG. 13(B). No other organ showed significantdifferences in weight across the three ketone precursors, as seen inFIG. 13(A).

Ketone supplementation effects on body weight

Rats were administered SO, MO, or SM daily for 28 days as describedabove. Each week the animals were weighed. Rats that were treated withSO, MO, or SM gained significantly less weight compared to controls, asseen in FIG. 14. The control animals continued to gain weight throughoutthe month-long study. In comparison, the ketone supplement treatmentgroups all gained less weight. A 2 to 3-fold reduction in weight gainwas noted in the SO and SM groups. A key finding of this study is thatthe SM group was able to limit weight gain in an unexpected way as theamounts of BHB and MCT when combined were much lower than the amounts ofthose ingredients used in the SO and MO treatment groups. This effect onbody weight gain is likely due to appetite suppression caused by thesupplements, rather than a reduced intake of the supplement itself, asforce feeding controlled for dosing.

Example 5

In a non-limiting example, a 16-ounce drink formula containing thefollowing ingredients is consumed by an individual three times a day.

TABLE 6 List of ingredients used in preparing a ketosis inducing drinkformula. Ingredient Amount Sodium beta-hydroxybutyrate 11 grams (1.9grams sodium, 9.1 grams beta-hydroxybutyrate) Potassiumbeta-hydroxybutyrate 7.1 grams (1.9 grams potassium, 5.2 gramsbeta-hydroxybutyrate) MCT oil, USP 30 grams Ancillary Ingredients (notused in tests below) Calcium beta-methyl-beta- 1000 milligramshydroxybutyrate Arginine alpha-ketoglutarate 1000 milligrams SodiumR-alpha lipoic acid 100 milligrams Thiamine 20 mg Riboflavin 20 mgNiacin 20 mg Pyridoxine 20 mg Ascorbic acid 750 mg Citric acid 1000 mgMalic acid 1000 mg Sodium benzoate 300 mg Potassium sorbate 300 mgAcesulfame K 10 mg Aspartame 30 mg Xanthan gum 1000 mg Flavoring —

The data presented herein supports the use of a unique combination ofbeta hydroxybutyrate and medium chain triglycerides in order to generatea unique blood ketone profile necessary for effectively enteringtherapeutic ketosis. There are at least two populations that wouldimmediately benefit from this combination formulation. First, childrenwith epilepsy benefit from ketosis as it has been shown to reduceseizures. Second, people that are looking to lose weight, suppress theappetite, and enhance physical performance benefit from ketosis toenhance their results. In both populations, adherence to a ketogenic,carbohydrate-restricted diet as a means of generating ketosis is limitedby the difficulty of generating blood ketone levels that aresufficiently elevated and prolonged. Slow entry into ketosis leads toprolonged side effects such as physical distress and mood depression.Additionally, even slight deviations from the ketogenic diet causepertubations in blood ketone levels, thereby quickly kicking the subjectout of therapeutic ketosis. The data presented herein show that thecombination of BHB and MCT results in a more rapid induction of ketosis,with greater peak levels of blood ketones, and longer sustainment ofketosis, compared to either component alone, thereby overcoming thedrawbacks of current approaches.

The combination of BHB and MCT is unique in that it also avoids thecomplications that arise from using either substance alone. Consumingsufficient amounts of BHB salts in order to quickly enter and maintainketosis results in sodium overload and electrolyte imbalances andtherefore is not practical. Consuming sufficient quantities of MCT inorder to quickly enter and maintain ketosis results in severegastrointestinal distress and therefore is also not practical. The datapresented herein shows that reduced amounts of both substances can beused in combination, and not only improves tolerability, but alsogenerates a unique blood ketone profile that has benefits not seen withthe individual components when used in isolation.

In the preceding specification, all documents, acts, or informationdisclosed does not constitute an admission that the document, act, orinformation of any combination thereof was publicly available, known tothe public, part of the general knowledge in the art, or was known to berelevant to solve any problem at the time of priority.

It is also to be understood that the following claims are intended tocover all of the generic and specific features of the invention hereindescribed, and all statements of the scope of the invention which, as amatter of language, might be said to fall therebetween.

What is claimed is:
 1. A composition for inducing ketosis, suppressingappetite, or promoting weight loss in a mammal, comprisingtherapeutically effective amounts of: at least one medium chain fattyacid or ester thereof; and a beta-hydroxybutyrate monomer salt mixturecomprising a plurality of beta-hydroxybutyrate monomer salts with atleast two beta-hydroxybutyrate monomer salts selected from the groupconsisting of sodium beta-hydroxybutyrate, potassiumbeta-hydroxybutyrate, calcium beta-hydroxybutyrate, magnesiumbeta-hydroxybutyrate, lithium beta-hydroxybutyrate, argininebeta-hydroxybutyrate, lysine beta-hydroxybutyrate, histidinebeta-hydroxybutyrate, ornithine beta-hydroxybutyrate, creatinebeta-hydroxybutyrate, agmatine beta-hydroxybutyrate, and citrullinebeta-hydroxybutyrate.
 2. The composition of claim 1, wherein thebeta-hydroxybutyrate monomer salt mixture comprises at least one ofsodium beta-hydroxybutyrate, potassium beta-hydroxybutyrate, calciumbeta-hydroxybutyrate, or magnesium beta-hydroxybutyrate.
 3. Thecomposition of claim 1, wherein the beta-hydroxybutyrate monomer saltmixture comprises at least two of sodium beta-hydroxybutyrate, potassiumbeta-hydroxybutyrate, calcium beta-hydroxybutyrate, or magnesiumbeta-hydroxybutyrate.
 4. The composition of claim 1, wherein thebeta-hydroxybutyrate monomer salt mixture comprises at least three ofsodium beta-hydroxybutyrate, potassium beta-hydroxybutyrate, calciumbeta-hydroxybutyrate, or magnesium beta-hydroxybutyrate.
 5. Thecomposition of claim 1, wherein the beta-hydroxybutyrate monomer saltmixture comprises: at least one of sodium beta-hydroxybutyrate,potassium beta-hydroxybutyrate, calcium beta-hydroxybutyrate, ormagnesium beta-hydroxybutyrate; and at least one of argininebeta-hydroxybutyrate, lysine beta-hydroxybutyrate, histidinebeta-hydroxybutyrate, ornithine beta-hydroxybutyrate, creatinebeta-hydroxybutyrate, agmatine beta-hydroxybutyrate, or citrullinebeta-hydroxybutyrate.
 6. The composition of claim 1, wherein thebeta-hydroxybutyrate monomer salt mixture comprises at least one ofarginine beta-hydroxybutyrate, lysine beta-hydroxybutyrate, histidinebeta-hydroxybutyrate, ornithine beta-hydroxybutyrate, creatinebeta-hydroxybutyrate, agmatine beta-hydroxybutyrate, or citrullinebeta-hydroxybutyrate.
 7. The composition of claim 1, wherein thebeta-hydroxybutyrate monomer salt mixture comprises a racemicDL-beta-hydroxybutyrate monomer salt.
 8. The composition of claim 1,further comprising at least one of 1,3-butanediol, ethyl acetoacetate,ethyl beta-hydroxybutyrate.
 9. The composition of claim 1, wherein theat least one medium chain fatty acid or ester thereof is selected fromthe group consisting of medium chain triglycerides, coconut oil, coconutmilk powder, fractionated coconut oil, palm kernel oil, isolatedhexanoic acid, isolated octanoic acid, isolated decanoic acid,ethoxylated triglycerides thereof, enone triglyceride derivativesthereof, aldehyde triglyceride derivatives thereof, monoglyceridederivatives thereof, diglyceride derivatives thereof, triglyceridederivatives thereof, alkyl esters thereof, and mixtures thereof.
 10. Thecomposition of claim 1, further comprising at least one additionalcomponent selected from the group consisting of amino acids, amino acidmetabolites, vitamins, minerals, coconut milk powder, electrolytes,NADH, tetrahydrobiopeterin, alpha-ketoglutaric acid, alpha lipoic acid,nutritional co-factors, calcium beta-methyl-beta-hydroxybutyrate,arginine alphaketoglutarate, sodium R-alpha lipoic acid, thiamine,riboflavin, niacin, pyridoxine, ascorbic acid, citric acid, malic acid,sodium benzoate, potassium sorbate, acesulfame K, aspartame, xanthangum, non-toxic minerals salts, and combinations thereof.
 11. Thecomposition of claim 1, wherein the composition is a dry powder.
 12. Acomposition for inducing ketosis, suppressing appetite, or promotingweight loss, comprising therapeutically effective amounts: at least onemedium chain fatty acid or ester thereof; and a beta-hydroxybutyratemonomer salt mixture comprising a plurality of beta-hydroxybutyratemonomer salts with at least two being sodium beta-hydroxybutyratemonomer salt, potassium beta-hydroxybutyrate monomer salt, calciumbeta-hydroxybutyrate monomer salt, or magnesium beta-hydroxybutyratemonomer salt.
 13. The composition of claim 12, wherein thebeta-hydroxybutyrate monomer salt mixture comprises at least twobeta-hydroxybutyrate monomer salts of sodium, potassium, calcium, ormagnesium.
 14. A method of inducing ketosis, suppressing appetite, orpromoting weight loss in a mammal, comprising administering thecomposition of claim 1 to a mammal.
 15. The method of claim 14, whereinadministering the composition comprises administering a daily amount ofabout 2 grams to about 50 grams of the beta-hydroxybutyrate monomer saltmixture.
 16. The method of claim 14, wherein administering thecomposition comprises administering a daily amount of about 5 grams toabout 30 grams of the beta-hydroxybutyrate monomer salt mixture.
 17. Themethod of claim 14, wherein administering the composition comprisesadministering a daily amount of about 10 grams to about 20 grams of thebeta-hydroxybutyrate monomer salt mixture.
 18. The method of claim 14,wherein the beta-hydroxybutyrate monomer salt mixture comprises at leasttwo of sodium beta-hydroxybutyrate, potassium beta-hydroxybutyrate,calcium beta-hydroxybutyrate, or magnesium beta-hydroxybutyrate.
 19. Themethod of claim 14, wherein the beta-hydroxybutyrate monomer saltmixture comprises at least three of sodium beta-hydroxybutyrate,potassium beta-hydroxybutyrate, calcium beta-hydroxybutyrate, ormagnesium beta-hydroxybutyrate.
 20. The method of claim 14, wherein themammal is a human.
 21. The method of claim 14, wherein administering thecomposition comprises administering a daily amount of about 5 grams toabout 50 grams of the at least one medium chain fatty acid or esterthereof.
 22. The method of claim 14, wherein administering thecomposition comprises administering a daily amount of about 10 grams toabout 40 grams of the at least one medium chain fatty acid or esterthereof.
 23. The method of claim 14, wherein administering thecomposition comprises administering a daily amount of about 15 grams toabout 30 grams of the at least one medium chain fatty acid or esterthereof.